Specific oligonucleotide primer pairs and probes for discriminating specific analytes

ABSTRACT

The present invention provides methods and apparatus for detecting and discriminating multiple analytes within a test sample which are simple, user-friendly, cost-effective and fast. In particular, it is preferred that the overall time for sample preparation, nucleic acid sequence amplification, and nucleic acid sequence differentiation be about 5 hours or less. The methods of the present invention comprise (i) rapid sample processing means for rapidly preparing sample material of various types for amplification of nucleic acid sequences using unique nucleic acid extraction buffer formulations, (ii) multianalyte non-preferential amplifying process means for simultaneously and non-preferentially amplifying multiple target nucleic acid sequences, if present within the sample, using appropriate primer oligonucleotides optimized to achieve substantially similar amplification efficiencies, and (iii) multianalyte recognition process means for detecting and discriminating amplified nucleic acid sequences which incorporate nucleic acid sequence mismatch detection means for differentiating minor mismatches between multiple amplified nucleic acid sequences, including only single base mismatches, using appropriate probe oligonucleotides modified with neutral base substitution molecules. The processing kit products in accord with the present invention may incorporate all, or only some, of the above-described means.

This application is a divisional of application Ser. No. 08/587,209,filed Jan. 16, 1996, U.S. Pat. No. 5,612,473.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to nucleic acid sequence based detectiontechnology. In particular, the present invention relates to methods andapparatus permitting the detection and discrimination of multipleanalytes within various types of sample material.

2. Background Information

Accurate detection of biological analytes present in various types oftest samples is useful for many purposes including clinical,experimental, and epidemiological analyses. Because the geneticinformation in all living organisms is carried largely in the nucleicacids, either double-stranded deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), detection and discrimination of specific nucleic acidsequences permits the presence, or absence, of a particular analytewithin a test sample to be determined.

The development of the polymerase chain reaction (PCR) process foramplifying one or more targeted nucleic acid sequences within a sampleor mixture of nucleic acid(s) has greatly facilitated processes fordetecting and discriminating specific nucleic acid sequences. See, e.g.,U.S. Pat. No. 4,965,188, the disclosure of which is herein incorporatedby reference. For each target nucleic acid sequence to be amplified, thePCR process involves treating separate complementary strands of nucleicacid with two primers selected to be substantially complementary toportions of the target nucleic acid sequence within the two strands. Theprimers are extended with a thermostable enzyme to form complementaryprimer extension products which, when separated into their complementarystrands, produce template strands for extending the complementary primerinto the target nucleic acid sequence. The target nucleic acidsequences, when separated into their complementary strands, also act astemplates for synthesis of additional target nucleic acid sequences. Thesteps of the PCR amplification process involve temperature cycling toeffect hybridization of primers and templates, promotion of enzymeactivity to enable synthesis of the primer extension products, andseparation of the strands of the hybrids formed to produce additionaltemplate strands including strands of the synthesized target nucleicacid sequences. Each cycle exponentially increases the quantity oftarget nucleic acid sequences synthesized.

PCR amplification has proven useful in numerous clinical applicationsincluding the detection and/or diagnosis of infectious diseases andpathological genomic abnormalities as well as DNA polymorphisms that maynot be associated with any pathological state. PCR amplification isparticularly useful in circumstances where the quantity of the targetednucleic acid is relatively small compared to other nucleic acids presentin a sample, where only a small amount of the targeted nucleic acid isavailable, where the detection technique has low sensitivity, or wheremore rapid detection is desirable. For example, infectious agents may beaccurately identified by detection of specific characteristic nucleicacid sequences. Examples of such infectious agents include bacteria suchas Salmonella, Shigella, Chlamydia, and Neisseria, viruses such as thehepatitis virus, and parasites such as the malaria-causing Plasmodium.Because a relatively small number of pathogenic organisms may be presentin a sample, the DNA extracted from these organisms typicallyconstitutes only a very small fraction of the total DNA in the sample.Specific amplification of the characteristic DNA sequences, if present,greatly enhances the sensitivity and specificity of the detection anddiscrimination processes.

In addition, genetic sequences indicative of genetic disorders such assickle cell anemia, α-thalassemia, β-thalassemia, and cystic fibrosiscan be amplified for detection. Detection of genes associated withdisease states such as insulin-dependent diabetes or certain cancers isalso useful. PCR amplification is particularly useful when the amount ofnucleic acid available for analysis is very small such as in theprenatal diagnosis of genetic disorders using DNA obtained from fetalcells. The PCR amplification process has also enhanced the detection anddiscrimination of genetic variants which represent different alleles as,for example, HLA typing useful for determining compatibility of tissuefor transplantation, disease susceptibility, and paternity.

PCR amplification is a powerful tool for the laboratory researcher. Theprocedures for preparing clinical samples to extract suitable nucleicacids or mixtures thereof, however, are typically difficult andtime-consuming. For example, HLA typing usually requires purifiedgenomic DNA as a template for the PCR process. Yet, it may be verydifficult to extract and/or purify target nucleic acids, if present, insome types of sample material. Thus, the usefulness of PCR is limited insome circumstances although recent advances have been made. For example,it has been reported that it is possible to perform PCR directly onsmall samples of washed blood cells or whole blood by subjecting thesamples to boiling in water for 10 minute before conducting PCR. Wu, L.,McCarthy, B. J., Kadushin, J. M., Nuss, C. E., A Simple and EconomicMethod for Directly Performing PCR on Washed Blood Cells or on WholeBlood, Transgenica, 1994: 1(1): 1-5. On the other hand, processing ofother types of clinical samples such as, for example, stool samples,sputum samples, clotted blood samples and others, continues to be timeconsuming and difficult.

There are many circumstances where it would be useful to simultaneouslydetect and discriminate between multiple target nucleic acid sequencespresent or potentially present within a test sample. For example, anaccurate diagnosis of an infectious disease may require determiningwhich, if any, of numerous possible infectious agents are present in aclinical sample. Generally, the sample must be divided and multiple PCRamplification procedures must be separately performed with differentprimers, if available, for the different potential target nucleic acidsequences. This approach is very laborious. In addition, each PCRamplification process performed with available primers may yield amixture of nucleic acids, resulting from the original template nucleicacid, the expected target nucleic acid sequence products, and variousbackground non-target nucleic acid sequence products.

Although as many as three analytes have been simultaneously amplified bysome methods, these methods also require difficult and time-consumingtest sample preparation steps. A particular problem encountered whenattempting to simultaneously amplify multiple targeted nucleic acidsequences is the phenomena of preferential amplification. Becausedifferent primers have different amplification efficiencies under thesimultaneous processing conditions, preferential amplification resultsin disproportionate amplification of one or more target nucleic acidsequences such that the quantity of the preferentially amplifiedsequence(s) greatly exceeds the quantity of the other amplifiedsequences present. Another problem encountered during simultaneousamplification of multiple analytes is cross-reactivity. Significantsequence matches between the different primers can diminishamplification efficiency of the specific target nucleic acid sequence orcause a false positive amplification to occur. Thus, both preferentialamplification and cross-reactivity must be prevented or minimized topermit accurate and efficient simultaneous analysis of multipleanalytes.

Numerous methods for detecting and discriminating nucleic acid sequencesusing oligonucleotide probes, i.e., probes complementary to thePCR-amplified products, are known. Typically, a solid phase system isused. For example, either the PCR amplified products or the probes maybe affixed directly onto a series of membranes. The non-affixedcomponents, i.e., either the probes or the PCR products, respectively,are then added to the separate membranes under hybridization conditions.Either the probes or the PCR products are labeled with some type oflabel moiety so as to be detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. Examples of labelmoieties include fluorescent dyes, electron-dense reagents, enzymescapable of depositing insoluble reaction products or of being detectedchromogenically, such as horseradish peroxidase or alkaline phosphatase,a radioactive label such as ³² p, or biotin. Hybridization between thePCR products and probes will occur only if the components aresufficiently complementary to each other. After hybridization, a washingprocess removes any non-hybridized molecules so that detection ofremaining labeled component indicates the presence of probe/targetnucleic acid hybrids.

In addition to the classic dot-blot detection methods, solid-phasesystems utilizing microtiter plates are also known. Various methods havebeen used to immobilize the desired component, either the probe or thePCR products, onto the microplate. In one approach, hydrophobic actionpassively adsorbs the component onto the microplate. Alternatively, thebiotin/avidin interaction is utilized by, for example, incorporatingbiotin onto the component and passively absorbing avidin molecules ontothe microplate such that the biotinylated components become bound to theavidin. A concern with these techniques, however, is that thehydrophobic binding used to immobilize the components to the plate isless efficient than desired. Only a relatively small quantity ofcomponent becomes bound to the plate and the hydrophobic binding may notwithstand stringent hybridization or washing procedures such that someof the bound components or probe/nucleic acid complexes may be washedaway during processing. The use of covalent linking chemistry hasrecently been shown to produce more consistent and efficient binding ofoligonucleotide probes to microplate plastic surfaces. Wu, L., Chaar,O., Bradley, K., Kadushin, J., HLA DR DNA Typing With a MicrotiterPlate-Based Hybridization Assay, Hum. Immun., 1993, 37: p. 141. In thistechnology, the oligonucleotide probes are attached covalently tochemical linkers on the plastic surface via the 5'-end phosphate group,amine group, or other reactive moiety. This approach does appear toimprove efficiency while simplifying the procedure.

For some purposes, it is necessary to identify variations in nucleicacid sequences such as single or multiple nucleotide substitutions,insertions or deletions. These nucleotide variations may be mutant orpolymorphic allele variations. Of particular interest and difficulty isthe discrimination of single-base mismatched nucleic acid sequences.Sequence-specific oligonucleotide probes, i.e., probes which are exactlycomplementary to an appropriate region of the target nucleic acidsequence, are typically used. All primers and probes, however, hybridizeto both exactly complementary nucleic acid sequence regions as well asto sequences which are sufficiently, but not exactly, complementary,i.e., regions which contain at least one mismatched base. Thus, aspecific probe will hybridize with the exact target nucleic acidsequence as well as any substantially similar but non-target nucleicacid sequences which are also present following the amplificationprocess.

Various approaches to discriminating these similar hybrids from oneanother have been used. For example, it is known that the hybridswherein the base-matching between the probe and nucleic acid sequence isexactly complementary, i.e., hybrids containing the exact targetsequence, are bound together more strongly than are hybrids wherein thebase matching is less than perfect. Accordingly, stringent washingconditions and/or processing with toxic chemicals are imposed to affectthe physical properties of the hybrid complexes and, in theory, causethe weaker complexes to disassociate. These known methods for detectingnucleic acid sequence base mismatches are too difficult, harsh, andinconvenient for routine laboratory use.

In view of the foregoing, it would be advantageous to provide methodsand apparatus for improving the efficiency and decreasing the timerequired to prepare and amplify multiple target nucleic acid sequenceswithin a test sample by permitting various types of sample material tobe prepared for DNA amplification without laborious nucleic acidextraction and purification steps.

It would be another advantage to provide methods and apparatus forimproving the efficiency and decreasing the time required to prepare andamplify multiple targeted nucleic acid sequences within a test sample bypermitting multiple target nucleic acid sequences within the sample tobe simultaneously and non-preferentially amplified.

It would be another advantage to provide such methods and apparatus forimproving the efficiency and decreasing the time required to prepare andamplify multiple targeted nucleic acid sequences within a test samplewhich are simple, user-friendly, cost-effective and fast.

It would be still another advantage to provide methods and apparatus fordetecting and discriminating even single-base mismatches betweenmultiple amplified nucleic acid sequences which do not requirestringent, harsh, and inconvenient processing conditions.

SUMMARY OF THE INVENTION

It is a primary object of the present invention to provide methods andapparatus for improving the efficiency of detection and discriminationof multiple analytes within a test sample by permitting various types ofsample material to be rapidly and simply prepared for nucleic acidamplification and by permitting multiple target nucleic acid sequenceswithin a sample to be simultaneously and non-preferentially amplified.

It is another object of the present invention to provide methods andapparatus for detecting and discriminating multiple analytes within atest sample which are simple, user-friendly, cost-effective and fast. Inparticular, it is preferred that the overall time for samplepreparation, nucleic acid sequence amplification, and nucleic acidsequence differentiation be about 5 hours or less.

It is an additional object of the present invention to provide suchmethods and apparatus which permit virtually any type of sample materialto be simply and rapidly prepared for a nucleic acid amplifying process.In particular, it is desired to provide rapid sample processing meansfor preparing a sample which avoids laborious nucleic acid extractionand purification steps. Preferably, the time for performance of therapid sample processing means is about 5 minutes or less.

It is still a further object of the present invention to provide methodsand apparatus for detecting and discriminating multiple analytes withina test sample which permit multiple target nucleic acids to besimultaneously amplified in a non-preferential manner. In particular, itis desired to provide rapid and efficient multianalyte non-preferentialamplifying process means for simultaneously and non-preferentiallyamplifying multiple target nucleic acid sequences, if present.Preferably, the time for performance of the multianalytenon-preferential amplifying process means is about 2 hours or less.

Yet another object of the present invention is to provide methods andapparatus for detecting and discriminating multiple analytes within atest sample which permits rapid and accurate detection anddiscrimination of target nucleic acid sequences. In particular, it isdesired to provide multianalyte recognition process means which aresimple, user-friendly, and rapid and which incorporate nucleic acidsequence mismatch detection means for discriminating between amplifiednucleic acid sequences having minor base mismatches, including only asingle base mismatch. Preferably, the time for performance of themultianalyte recognition process means, including nucleic acid sequencemismatch detection, if necessary, is about 2 hours or less.

These and other objects and advantages of the invention will be betterunderstood by reference to the detailed description, or will beappreciated by the practice of the invention. To achieve the foregoingobjects, and in accordance with the invention as embodied and broadlydescribed herein, the methods of the present invention comprise (i)rapid sample processing means for rapidly preparing sample material ofvarious types for amplification of nucleic acid sequences using uniquenucleic acid extraction buffer formulations, (ii) multianalytenon-preferential amplifying process means for simultaneously andnon-preferentially amplifying multiple target nucleic acid sequences, ifpresent within the sample, using appropriate primer oligonucleotidesoptimized to achieve substantially similar amplification efficiencies,and (iii) multianalyte recognition process means for detecting anddiscriminating amplified nucleic acid sequences which incorporatenucleic acid sequence mismatch detection means for differentiating minormismatches between multiple amplified nucleic acid sequences, includingonly single base mismatches, rising appropriate probe oligonucleotidesmodified with neutral base substitution molecules.

The unique extraction buffer formulation of the rapid sample processingmeans is effective with substantially any type of sample material.Preferably, the time for performance of the sample processing means isabout 5 minutes or less. The unique oligonucleotide primers of themultianalyte non-preferential amplifying process means are adapted toensure that cross-reactivity is avoided and that amplificationefficiency is substantially equal at the selected process conditions.Preferably, the time for performance of the multianalytenon-preferential amplifying process is about 2 hours or less.Preferably, the time for performance of the multiple analyte recognitionprocess, including nucleic acid sequence mismatch detection, ifnecessary, is about 2 hours or less.

The apparatus of the present invention comprise processing kit productscomprising means for (i) rapidly preparing virtually any type of samplematerial, using appropriate reagents, for nucleic acid sequenceamplification, (ii) simultaneously and non-preferentially amplifyingmultiple target nucleic acid sequences, if present within the sample,using appropriate primer oligonucleotides, and (iii) detecting anddiscriminating multiple amplified nucleic acid sequences, includingnucleic acid sequences having only single-base mismatches, usingappropriate probe oligonucleotides. The processing kit products inaccord with the present invention may incorporate all, or only some, ofthe above-described means.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to methods and apparatus useful fordetecting and discriminating multiple analytes potentially presentwithin various types of sample materials. In particular, the methods andapparatus of the present invention utilize rapid sample processing meansfor preparing virtually any type of sample material for amplification ofnucleic acid sequences contained therein, multianalyte non-preferentialamplifying process means for simultaneously and non-preferentiallyamplifying multiple target nucleic acid sequences, if present, withinthe sample, and multianalyte recognition process means which incorporatenucleic acid sequence mismatch detection means for detecting anddiscriminating minor base mismatches, including single-base mismatches,between multiple amplified nucleic acid sequences.

The methods and apparatus in accord with the present invention permitdetection and differentiation of multiple analytes within a sample to beperformed within a relatively short time period, preferably less thanabout 5 hours. It is most preferred that the time for sample preparationbe about 5 minutes or less, the time for simultaneous non-preferentialamplification of the targeted nucleic acid sequences be about 2 hours orless, and the time for recognition of the multiple amplified nucleicacid sequences be about 2 hours or less.

The rapid sample processing means for preparing a sample foramplification of multiple nucleic acid sequences uses unique nucleicacid extraction buffer formulations effective with substantially anytype of sample material. The multianalyte non-preferential amplifyingprocess means utilizes multiple appropriate primer oligonucleotidesoptimized to ensure that cross-reactivity is avoided and thatamplification efficiency is substantially equal at the selected processconditions. The multianalyte recognition process is performed withappropriate probe oligonucleotides and is preferably performed onmicrotiter plates incorporating covalent linking technology to enhancethe binding efficiency of the probe/nucleic acid sequence hybrids. Inaddition, nucleic acid sequence mismatch detection means are provided topermit discrimination between amplified nucleic acid sequences havingminor mismatches, including only single base mismatches. The nucleicacid sequence mismatch detection means utilize appropriate probeoligonucleotides modified by at least one neutral base substitution anddo not require stringent or harsh processing conditions.

The apparatus of the present invention comprise processing kit productsfor detecting and discriminating multiple analytes within a test sample.The kit products comprise any or all of the following: (i) means forrapidly preparing virtually any type of sample material, usingappropriate reagents, for nucleic acid sequence amplification, (ii)means for simultaneously and non-preferentially amplifying multipletarget nucleic acid sequences, if present within the sample, usingappropriate primer oligonucleotides, and (iii) means for detecting anddiscriminating multiple amplified nucleic acid sequences, includingnucleic acid sequences having minor mismatches and even single basemismatches, using appropriate probe oligonucleotides.

One of the most important advantages of the methods and apparatus of thepresent invention is the time savings realized. The use of uniquelyformulated extraction buffer solutions permits very rapid preparation ofvirtually any type of sample material. The use of uniquely optimizedoligonucleotide primers ensures that preferential amplification isavoided and dramatically reduces the processing time for amplificationof the target nucleic acid sequences by permitting amplification ofmultiple target nucleic acid sequences to proceed simultaneously. Inaccord with the present invention, as many as six different targetnucleic acid sequences, and possibly more, can be non-preferentiallyamplified in one sample. Thus, a tremendous amount of time previouslyspent in preparing different types of sample materials and performingrepetitive amplification processes on separate sample portions is savedin accord with the present invention. In addition, the unique probesused in the multianalyte recognition process may incorporate neutralbase substitution means to facilitate accurate discrimination of nucleicacid sequences, including sequences having only a single basedifference, even under simple and user-friendly processing conditions.

The methods and apparatus of the present invention are extremelyversatile and have broad-reaching applications. For example,simultaneous detection of and discrimination between multiplepotentially present microorganisms in biological samples permits rapiddiagnosis of infections. It would also be useful to rapidly detect anddiscriminate between multiple potentially present microorganisms inother types of samples, such as foodstuffs, for various purposes such asdiagnostic/forensic or quality control purposes. Applications in geneticresearch are also numerous and versatile. The detection anddiscrimination of specific genetic sequences, including discriminationbetween single base mismatched sequences, for purposes includingdiagnosing genetic disorders or identifying genetic variances, arerapidly, simply, and accurately performed in accord with the presentinvention.

The methods and apparatus of the present invention will be describedboth generally and with reference to a specific clinical application.One exemplary clinical application of the methods and apparatus of thepresent invention is the detection and discrimination among multiplepotential infectious pathogens. In particular, the advantageousapplication of the methods and apparatus of the present invention in aGastroenteritis Panel for assisting in diagnosing the cause of acutegastroenteritis through stool sample analysis will be described.

There can be many causes of acute gastroenteritis. It is often importantto differentiate acute gastroenteritis from other conditions which maypresent similar signs and symptoms. In addition, it can be critical insome cases of infectious etiology to determine the specific causativeagent. In some circumstances, rapid initiation of specific and effectivepharmacologic therapy may be life-saving while, in other circumstances,use of inappropriate antimicrobial agents can actually be detrimental tothe patient. Five groups of potential infectious etiological agents forwhich a rapid detection and discrimination analysis would beparticularly helpful are Salmonella species, Shigella species andenteroinvasive Escherichia coli, Campylobacter species,enterohemorrhagic E. coli (particularly E. coli O157:H7), and Yersiniaenterocolitica. Selected characteristics of these five groups aredetailed below:

1. Salmonella Species

Disease: Mild gastroenteritis to life threatening typhoid fever,bacteremia, meningitis, respiratory disease, cardiac disease and boneinfections. Asymptomatic carrier state common.

Prevalence: Three million cases in U.S. estimated each year. Foodborneinfection common.

Diagnosis: Screened for using differential and moderately selectiveagar. Identified using biochemical battery and serotyping.

Time to Preliminary Test Results: 18-48 hours

Treatment: Antibiotic therapy appropriate only in certain clinicalcircumstances--may be detrimental in other circumstances. Antibioticresistance is common.

2. Shigella Species and Enteroinvasive E. coli (EIEC)

Disease: Responsible for most cases of bacillary dysentery--waterydiarrhea with blood and mucus. Also associated with systemic infectionsleading to death.

Prevalence: Most highly communicable of the bacterial diarrheas.Implicated in between 10 and 40 percent of diarrhea cases throughout theworld. Often responsible for outbreaks in day-care centers.

Diagnosis: Screened for using differential and moderately selectiveagar. Identified using biochemical battery and/or serotyping.

Time to Preliminary Test Results: 18-48 hours

Treatment: Often resistant to sulfonamides, tetracycline, ampicillin,and trimethoprim-sulfamethoxazole.

3. Campylobacter Species

Disease: One of the most common causes of sporadic bacterial enteritisin the U.S. Systemic infections may occur. May mimic acute appendicitisresulting in unnecessary surgery. Guillain-Barre syndrome is stronglyassociated with infection.

Prevalence: Over 2 million cases estimated to occur annually indeveloped nations. Many orders of magnitude higher in developingcountries. Animal hosts common.

Diagnosis: Usually requires microaerobic atmosphere and elevatedtemperature. Use selective media containing antimicrobials to inhibitnormal flora. Identify using Gram stain and oxidase test. Can be sent toreference laboratory if further identification is needed.

Time to Preliminary Test Results: 48-72 hours

Treatment: Erytromycin and ciprofloxacin are usually drugs of choice.

4. Enterohemorrhagic E. coli (EHEC) especially strain O157:H7

Disease: Associated with bloody and nonbloody diarrhea (hemorrhagiccolitis), hemolytic-uremic syndrome (HUS--a major cause of acute renalfailure in children), thrombotic thrombocytopenic purpura, and death.

Prevalence: E. coli O157:H7 is estimated to cause more than 20,000infections and as many as 250 deaths each year in. U.S. Commonlyassociated with undercooked hamburger.

Diagnosis: Does not ferment sorbitol rapidly--screen by usingsorbitol-MacConkey agar (SMAC) plate. Assay for O157 antigen withantiserum. Send out to reference laboratory for H7 confirmation.

Time to Preliminary Test Results: 18-48 hours

Treatment: Antibiotic treatment may make disease worse.

5. Yersinia enterocolitica

Disease: A major cause of human gastroenteritis that can lead to serioussystemic infections. Fatal cases of yersiniosis caused by transfusion ofinfected blood have been reported in several countries.

Prevalence: Becoming increasingly important in U.S., Canada, andnorthern Europe. Commonly associated with foodborne epidemics.

Diagnosis: Cultured at lower temperatures using selective medium such asCIN agar (cefsulodin-irgasan-novobiocin agar). Identified usingbiochemical batteries. Serologic assays may be useful.

Time to Preliminary Test Results: 24 hours to 7 days

Treatment: Use of aminoglycosides and/or trimethoprim-sulfamethoxazoleindicated in extra-intestinal cases.

1. RAPID SAMPLE PROCESSING MEANS

It will be appreciated that the rapid detection and discrimination ofthe above potential pathogens would be extremely useful. Because acutegastroenteritis is localized in the gastrointestinal tract, the mostappropriate sample material for analysis of potential pathogens is stoolsample material. Prior art methods of analyzing stool sample materialtypically involve time-consuming and laborious handling and preparationprocesses. Detection and discrimination of pathogens present, if any, instool sample material is typically challenging for the clinicallaboratory. In accord with the methods and apparatus of the presentinvention, however, stool sample material is rapidly and simply preparedfor subsequent nucleic acid sequence amplification, detection, anddiscrimination.

In accord with the present invention, it is desired to provide methodsand apparatus which permit virtually any type of sample material to besimply and rapidly prepared for a nucleic acid amplifying process.Conventional techniques for extracting and/or purifying nucleic acids invarious types of sample materials are generally time-consuming andlabor-intensive. In addition, it is very difficult, or impractical, toextract and/or purify nucleic acids within certain types of samplematerials such as, for example, stool samples, blood samples containingdifferent anticoagulants, or clotted blood samples. The presentinvention provides rapid sample processing means for preparing a samplefor nucleic acid sequence amplification which avoids laborious nucleicacid extraction and purification steps. Preferably, the time forperformance of the sample processing means is about 5 minutes or less.

The rapid sample processing means of the present invention utilizeunique extraction buffer solutions to effect nucleic acid extraction invirtually any type of sample material. Known extraction buffer solutionstypically comprise a buffer such as Tris-HCl (available from SigmaChemical Co., St. Louis, Mo., USA), EDTA (ethylenediaminetetraaceticacid), and at least one detergent composition. In contrast, theinventive extraction buffer solutions comprise Tris-HCl, EDTA, at leastone detergent, and at least one type of salt in a molar concentration,preferably in the range of 1M to 6M. Preferably, the inventiveextraction buffer solution comprises two detergents such as, forexample, Igepal CA 630 and Tween 20 (both available from Sigma ChemicalCo., St. Louis, Mo., USA), and about 2.0M NaCl.

Example 1 compares the effectiveness of the inventive rapid sampleprocessing means with two different conventional sample processing meansfor preparing stool samples for a nucleic acid amplification processreferred to as phenol/chloroform extraction and protein salting-out.Performance time for the rapid sample processing means was approximately3.5 minutes. In striking contrast, the performance time for thephenol/chloroform extraction was approximately 4.5 hours and theperformance time for the protein salting-out method was approximately12.75 hours. Example 2 illustrates the effectiveness of the inventiverapid sample processing means for sample materials other than stoolsamples.

EXAMPLE 1 MATERIALS AND METHODS I. SAMPLES

Clinical stool specimens were obtained from Utah State HealthLaboratory, Salt Lake City, Utah; Medical University of South Carolina,Charleston, S.C.; Primary Children's Medical Center, Salt Lake City,Utah; and Laboratory Corporation of America, Salt Lake City, Utah.Pathogens were isolated by agar plate culture in all laboratories. Allpathogenic isolates from specimens obtained from Laboratory Corporationof America were confirmed by Utah State Health Laboratory.

II. DNA EXTRACTION FROM CLINICAL STOOL SAMPLES

A. Rapid Sample Processing Means (Approximate time: 3.5 minutes)

A 5% solution of well mixed clinical sample was prepared in a 2 mlmicrocentrifuge tube with 1000 μl of a preferred extraction buffersolution of the present invention comprising the following: 1% IgepalCA630, 0.5% Tween 20, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 2M NaCl(all chemicals obtained from Sigma Chemical Co., St. Louis Mo., USA).The mixture was strongly vortexed. The tube was centrifuged for 5seconds at 16,000×g in an Eppendorf 5415 C microcentrifuge (BrinkmanInstruments, Inc., Westbury, N.Y., USA). Eight hundred μl supernatantwas transferred to a 2 ml microwaveable screw-cap centrifuge tube(Sarstadt, Newton, N.C., USA) and microwaved in a Kenmore 89250microwave (Sears, Salt Lake City, Utah, USA) on high (˜750 Watts) for 15seconds, mixed, and microwaved 10 more seconds. After microwaving, thetube was centrifuged for 1 minute at 16,000×g to pellet precipitatedprotein. Without disturbing the pelletted material, a measured amount ofsupernatant was transferred to a new microcentrifuge tube, containing anequal volume of room temperature isopropanol (Sigma Chemical Co.) andmixed by vortexing. The tube was centrifuged for 1 minute at 16,000×g topellet DNA. Supernatant was decanted and 1 ml 70% ethanol was added topellet. Tube contents were vortexed and tube was centrifuged at 16,000×gfor 1 minute. Without disturbing pellet, supernatant was removed byaspiration and 35 μl TE buffer (10 mM Tris-HCl, ph 8.0, 1 mM EDTA) wasadded to dissolve DNA.

Prepared samples were frozen at -20 degrees Celsius (°C.) untilquantitation, purity and polymerase chain reaction (PCR) analyses wereperformed.

B. Phenol/chloroform extraction (Approximate time: 4.5 hours)

A 5% solution of well mixed clinical stool sample was prepared in 1000μl PBS, and mixed well by vortexing. The solution was centrifuged at1750×g for 1 minute in an Eppendorf 5415 C rmicrocentrifuge (BrinkmanInstruments, Inc., Westbury, N.Y., USA) to pellet insoluble particulatematerial. Supernatant was transferred to a new microcentrifuge tube andcentrifuged at 16,000×g for 5 minutes to pellet bacteria Supernatant wasremoved and the pellet was resuspended in 100 μl phosphate bufferedsaline (PBS), pH 7.4. Fifty μl Digestion/Lysis Buffer (50 mM Tris-HCl,pH 8.0, 100 mM EDTA, 100 mM NaCl, 3% sodium dodecyl sulfate (SDS),2mg/ml Proteinase K)(all chemicals obtained from Sigma Chemical Co.) wasadded to the tube and gently vortexed to mix. Sample was incubated for2.5 hours at 55° C. and vortexed gently every 30 minutes.Phenol/Chloroform/Isoamyl extraction was performed as follows: An equalvolume of Phenol/Chloroform/Isoamyl alcohol (25:24:1)(Sigma ChemicalCo.) was added to the tube and mixed by inverting tubes 2-3 times. Theorganic and aqueous phases were separated by centrifugation at 1750×gfor 15 minutes. The aqueous layer was transferred to a newmicrocentrifuge tube. The Phenol/Chloroform/Isoamyl extraction wasperformed three more times. Next, the same extraction procedure wasperformed twice more with Chloroform/Isoamyl alcohol (24:1)(SigmaChemical Co.). From the final extraction, the aqueous layer wastransferred to a new tube and DNA was precipitated by adding an equalvolume of room temperature isopropanol. The DNA was collected bycentrifuging for 5 minutes at 16,000×g. The supernatant was removed andthe DNA pellet was air dried for 15 minutes then resuspended in 35 μl TEbuffer (10 mM Tris-HCl, ph 8.0, 1 mM EDTA) was added to dissolve DNA.

Prepared samples were frozen at -20° C. until quantitation, purity andpolymerase chain reaction (PCR) analyses were performed.

C. Protein salting-out method (Approximate time: 12.75 hours)

A 5% solution of well mixed clinical stool sample was prepared in 1000μl PBS, and vortexed well. The solution was centrifuged at 1750×g for 1minute in an Eppendorf 5415 C microcentrifuge (Brinkman Instruments,Inc., Westbury, N.Y., USA) to pellet insoluble particulate material.Supernatant was transferred to a new microcentrifuge tube andcentrifuged at 16,000×g for 5 minutes to pellet bacteria. Supernatantwas removed and the pellet was resuspended in 300 μl Nuclei Lysis Buffer(10 mM Tris-HCl, pH 8.2, 400 mM NaCl, 2 mM EDTA)(all chemicals obtainedfrom Sigma Chemical Co.) Lysate was digested overnight at 37° C. with 50μl Digestion Solution (1% SDS, 2 mM EDTA, 1 mg/ml Proteinase K)(allchemicals obtained from Sigma Chemical Co.) and 20 μl 10% SDS (SigmaChemical Co.). One hundred μl of 6M NaCl was added to the sample and thetube was vigorously shaken for 15 seconds, then centrifuged for 15minutes at 1750×g. A measured amount of supernatant was transferred to anew tube. Exactly 2 volumes of room temperature absolute ethanol(Spectrum Chemical Mfg. Corp., Gardena, Calif., USA) was added and thetube was inverted several times. DNA was pelleted by centrifuging at16,000×g for 15 minutes. Supernatant was removed by aspiration and 35 μlTE buffer (10 mM Tris-HCl, ph 8.0, 1.0 mM EDTA) was added to dissolveDNA.

Prepared samples were frozen at -20° C. until quantitation, purity andpolymerase chain reaction (PCR) analyses were performed.

III. SPECTROPHOTOMETRIC QUANTITATION AND PURITY ANALYSIS

All DNA extractions were diluted 1:50 in purified water and absorbancewas read at 260 nm and at 280 nm using a Hitachi U-3000Spectrophotometer (Tokyo, Japan). Absorbance of the sample at 260 nm wasused to calculate DNA concentration. The ratio of the absorbancereadings at 260 nm and at 280 nm was used to determine purity of DNA inthe sample. Because the quantities of PCR products were small, however,the sensitivity limits of the detection equipment were approached.

IV. PCR AND GEL DETECTION

For amplification, a 50 μl reaction mixture was prepared using 5 μl of10 X PCR buffer (Promega Corp., Madison, Wis., USA), 1 μl (5.0 U/μl) Taqpolymerase, 1 μl (10 mM) each DNTP (Promega Corp., Madison, Wis., USA),5 μl (400 ng total primer concentration) primers, 30 μl molecular gradewater, and 5 μl (200 ng or less total DNA concentration) sample. Primeroligonucleotides, specific for the particular target nucleic acidsequences known to be present in the clinical samples were synthesizedby Genosys Biotechnologies, Inc. (Woodlands, Tex., USA), Integrated DNATechnologies, Inc. (Coralville, Iowa, USA), Genemed Biotechnologies,Inc. (San Francisco, Calif., USA), and Cruachem, Inc. (Dulles, Va.,USA).

PCR amplification was performed using a Perkin Elmer 9600 thermocycler(Norwalk, Conn., USA) with one denaturing cycle for 3 minutes at 96° C.followed by 35 cycles as follows: 30 seconds at 94° C. for denaturing,20 seconds at 61° C. for annealing, 20 seconds at 72° C. for extension.

PCR products were visualized (and confirmed by size) by ethidium bromidestaining after electrophoresis in 4% Nu Sieve 3:1 agar (FMC, Rockland,Md., USA). A 50-1000 bp ladder (Bio Ventures, Inc., Murfreesboro, Tenn.,USA) was used as a molecular size marker.

RESULTS RAPID SAMPLE PROCESSING MEANS PROTOCOL DEVELOPMENT

A. Microwave time

Microwave time was examined from 10 seconds to 180 seconds using 10%stool samples diluted two-fold (undilute to 1:1024) in 200 μl of thepreferred extraction buffer solution of the present invention comprisingthe following: 1% Igepal CA63 0, 0.5% Tween 20, 10 mM Tris-HCl, pH 8.0,1 mM EDTA, and 2M NaCl (all chemicals obtained from Sigma Chemical Co.,St. Louis Mo., USA). Buffer solution temperature was measured using aTegam Model 821 Microprocessor Thermometer (Salt Lake City, Utah, USA)after microwaving was completed. The following results were obtained:

                                      TABLE 1                                     __________________________________________________________________________    Temperatures in Degrees Celsius                                               Micro-                                                                        wave                                                                              Dilutions:.                                                               Time                                                                              Non-                                                                      (sec)                                                                             dilute                                                                           1:2                                                                              1:4                                                                              1:8                                                                              1:16                                                                             1:32                                                                             1:64                                                                             1:128                                                                            1:256                                                                            1:512                                                                            1:1024                                      __________________________________________________________________________    10  48.2                                                                             54.0                                                                             46.4                                                                             43.9                                                                             44.7                                                                             47.5                                                                             42.9                                                                             ND ND ND 43.4                                        20  80.8                                                                             81.2                                                                             68.3                                                                             70.2                                                                             73.6                                                                             72.6                                                                             73.4                                                                             ND ND ND 69.9                                        30  90.6                                                                             91.6                                                                             91.2                                                                             80.1                                                                             87.5                                                                             ND ND ND ND ND ND                                          40  95.3                                                                             98.2                                                                             94.8                                                                             91.1                                                                             93.7                                                                             ND ND ND ND ND ND                                          50  ˜dry                                                                       93.7                                                                             98.0                                                                             96.7                                                                             95.0                                                                             ND ND ND ND ND ND                                          60  dry                                                                              dry                                                                              97.8                                                                             94.4                                                                             92.1                                                                             ND ND ND ND ND ND                                          90  dry                                                                              dry                                                                              dry                                                                              dry                                                                              dry                                                                              98.5                                                                             93.7                                                                             ND ND ND ND                                          180 dry                                                                              dry                                                                              dry                                                                              dry                                                                              dry                                                                              dry                                                                              dry                                                                              98.3                                                                             97.2                                                                             95.3                                                                             98.1                                        __________________________________________________________________________     ND = not done                                                            

Optimal microwave time of 20-30 seconds at high power (˜750 Watts) wasdetermined at a point where boiling temperatures were approximately metand where the sample did not lose appreciable volume or boil over. Themicrowave time was split during sample processing to optimize sampletemperature and to lessen sample boil-over or explosion. A total of15,000-22,500 Watt-seconds is preferred.

B. Centrifuge Time

The time periods used to centrifuge samples during precipitation ofprotein and precipitation of DNA were examined at 1, 3, 5, 10, and 15minutes. There were no marked differences in sample purity or DNA yield.

C. Ethanol vs. Isopropanol Precipitation

Absolute ethanol was compared to isopropanol for DNA precipitation. Itwas found that the use of isopropanol to precipitate DNA resulted inproduction of equal or greater quantities of PCR product duringamplification, as visualized on ethidium bromide stained agarose gels,than the use of absolute ethanol. The use of isopropanol also generallyresulted in equal or better absorbance ratios of extracted samples whencompared to absolute ethanol. Isopropanol is easier to obtain thanabsolute ethanol. Also, only one (sample) volume of isopropanol is usedto precipitate DNA compared to two (sample) volumes of absolute ethanol.Thus, isopropanol is more cost effective and preferred in the presentinvention.

II. CLINICAL SAMPLE ANALYSIS

A. Ratio of A260 nm/A280 nm

Spectrophotometric quantitation and purity analyses were performed asdescribed above on nine clinical samples extracted according to therapid sample processing means in accord with the present invention(method A) and nine samples prepared according to each of theconventional extraction methods, phenol/chloroform extraction (method B)and protein salting-out (method C). As shown in Table 2, the samplesextracted with the rapid sample processing means of the presentinvention demonstrated comparable A260/A280 ratios to samples extractedwith phenol/chloroform and better ratios than samples prepared by theprotein salting-out method.

                  TABLE 2                                                         ______________________________________                                        A260/A280 Ratios                                                              Method                                                                              1      2     3    4   5    6   7   8   9   Avg. S.D.                    ______________________________________                                        A     1.3    1.3   1.6  1.3 1.5  1.3 1.5 1.5 1.7 1.4  0.15                    B     1.0    1.2   2.0  1.4 1.7  1.9 1.6 17  1.5 1.6  0.32                    C     1.2    1.5   1.4  1.4 1.4  1.4 1.4 1.1 1.1 1.3  0.15                    ______________________________________                                    

B. DNA Amplification and Gel Detection

PCR amplification and gel detection was performed as described above onnine clinical samples extracted according to the rapid sample processingmeans in accord with the present invention (method A) and nine samplesprepared according to each of the conventional extraction methods,phenol/chloroform extraction (method B) and protein salting-out (methodC). The samples extracted with the rapid sample processing means of thepresent invention demonstrated equal amplification efficiency whencompared to samples subjected to phenol/chloroform extraction and betteramplification efficiency than samples prepared according to the proteinsalting-out method.

EXAMPLE 2

The rapid sample processing means of the present invention constitutes aprotein salting-out process accomplished by use of unique extractionbuffer solutions. A preferred extraction buffer solution contains 1%Igepal CA-630, 0.5% Tween 20, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 2MNaCl. The rapid sample processing means of the present invention weredeveloped for use with stool samples which are known to be one of themost difficult types of samples to prepare for nucleic acidamplification. The effectiveness of the rapid sample processing meansfor preparing stool samples is demonstrated in Example 1. In addition,the effectiveness of the rapid sample processing means in accord withthe present invention for preparing other sample types including clottedblood samples, blood samples collected with different anticoagulants,sputum samples, tissue culture cells, and bacterial cultures has alsobeen demonstrated.

For example, blood samples were collected from seven donors in NaCitrate, EDTA, heparin, and SST (clot) tubes. All samples were extractedin two trials using the method of the present invention. PCRamplification and gel detection demonstrated that all of these bloodsamples, regardless of collection type, amplified with β-globin primers.Similar results were obtained with sputum samples. Seven sputum samples,submitted for routine culture and/or Gram's stain to the Microbiologylaboratory at Veterans Affairs Medical Center, Salt Lake City, Utah,were extracted using the method of the present invention and all ofthese samples amplified with β-globin primers.

2. MULTIANALYTE NON-PREFERENTIAL AMPLIFYING PROCESS MEANS

As reviewed in the background section, amplification of target nucleicacid sequences is utilized to facilitate detection and discrimination.Although there are several known techniques for nucleic acid sequenceamplification, the polymerase chain reaction (PCR) amplification processhas proven particularly useful in numerous clinical applications.Amplification of multiple analytes potentially present within a sampleis generally accomplished by dividing the sample and performing multipleseparate PCR amplification procedures on separate sample portions, eachamplification procedure using a different primer pair specific for oneof the different potential target nucleic acid sequences. It would beadvantageous if multiple analytes could be amplified simultaneously.There are many circumstances where it would be useful to simultaneouslydetect and discriminate between multiple target nucleic acid sequencespresent or potentially present within a test sample. For example, anaccurate diagnosis of an infectious disease may require determiningwhich, if any, of numerous possible infectious agents are present in aclinical sample.

A particular problem encountered when attempting to simultaneouslyamplify multiple targeted nucleic acid sequences is the phenomena ofpreferential amplification. When attempting to simultaneously amplifymultiple analytes, the primer pairs intended to amplify the differenttarget nucleic acid sequences necessarily differ from each other.Different primers having different physical properties will havedifferent amplification efficiencies under the selected simultaneous PCRprocess conditions. For example, it is known that primers having ahigher content of guanosine and cytosine nucleotide bases, which formtriple rather than double bonds, tend to anneal better during PCR. Otherphysical properties which affect the amplification efficiency includethe primer melting temperature, the primer length, and the presence ofhairpin loops or dimers. Process conditions which affect theamplification efficiency include the magnesium concentration, theparticular polymerase enzyme employed, the enzyme concentration, theannealing temperature, and the primer concentration.

Preferential amplification results in disproportionate amplification ofone or more target nucleic acid sequences such that the quantity of thepreferentially amplified sequence(s) greatly exceeds the quantity of theother amplified sequences present. Another problem encountered duringsimultaneous amplification of multiple analytes is cross-reactivityamong the primers. Significant sequence matches between the differentprimers can diminish amplification efficiency of the target nucleic acidsequence or cause a false positive amplification to occur. Thus, bothpreferential amplification and cross-reactivity must be prevented orminimized to permit accurate and efficient simultaneous analysis ofmultiple analytes.

The multianalyte nucleic acid sequence amplifying process of the presentinvention permits non-preferential amplification of multiple analyteswithin a sample to proceed simultaneously. The multianalytenon-preferential amplifying process means employs unique primeroligonucleotides optimized to ensure that cross-reactivity is avoidedand that amplification efficiency is substantially equal at the selectedprocess conditions. Preferably, the time for performance of themultianalyte non-preferential amplifying process is about 2.0 hours orless.

As illustrated in Example 3, for the gastroenteritis panel application,there are preferably six potential target nucleic acid sequences. Fiveare the potential pathogens, Salmonella, Shigella, E. coli,Campylobacter, and Yersinia microorganisms. A sixth is a control gene,β-globin, which should be present in every properly prepared sample.

EXAMPLE 3 MATERIALS AND METHODS I. BACTERIAL STANDARDS

DNA for bacterial standards was extracted from lyophilized American TypeCulture Collection (ATCC) cultures, listed in Table 3, as follows: 300μl of extraction buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% TritonX-100, 0.5% Tween 20)(all chemicals obtained from Sigma Chemicals, St.Louis, Mo., USA) was added to each lyophilized vial. A 100 μl aliquotwas microwaved for 3 minutes on the high setting (˜750-775 watts). TheDNA concentration was estimated spectrophotometrically then diluted to aworking concentration of approximately 40 μg/ml.

                  TABLE 3                                                         ______________________________________                                        ATCC STANDARD STRAINS                                                         BACTERIA        ATCC NUMBER                                                   ______________________________________                                        Salmonella typhimurium                                                                        29946                                                         Shigella boydii 29929, 9207, 8702, 9210, 8764, 12027,                                         9905, 12028, 49812, 12030, 12031,                                             12033, 12032, 12035, 12034                                    Shigella dysenteriae                                                                          9361                                                          Shigella flexneri                                                                             29903                                                         Shigella sonnei 29930, 29029, 29030, 29031, 25931                             Campylobacter coli                                                                            33559                                                         Campylobacter feacalis                                                                        33709                                                         Campylobacter fetus                                                                           19438, 27374                                                  Campylobacter jejuni                                                                          29428, 43431                                                  Campylobacter lari                                                                            35221                                                         Campylobacter mucosalis                                                                       43264                                                         Escherichia coli O157:H7                                                                      43894, 35150                                                  Escherichia coli O25:K98:NM                                                                   43886                                                         Yersinia enterocolitica                                                                       23715, 27729, 29913, 9610                                     ______________________________________                                    

The DNA sequences for the target regions of amplification for the sixanalytes are known and can be accessed from the Genbank with thenucleotide sequence accession numbers shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        NUCLEOTIDE SEQUENCE ACCESSION NUMBERS                                         ______________________________________                                        Salmonella    Genbank #M90846                                                 Campylobacter Genbank #J05635                                                 Shigella/Enteroinvasive                                                                     Genbank #X16661                                                 E. Coli                                                                       Yersinia      Genbank #M29945                                                 Escherichia coli                                                                            Genbank #M14641                                                 β globin Genbank #J00179, #J00093, #J00094,                                            #J00096, #J00158, #J00159, #J00160,                                           #J00161, #J00162, #J00163, #J00164,                                           #J00165, #J00166, #J00167, #J00168,                                           #J00169, #J00170, #J00171, #J00172,                                           #J00173, #J00174, #J00175, #J00177,                                           #J00178, #K01239, #K01890, #K02544,                                           #M18047, #M19067, #M24868,                                                    #M24886, #X00423, #X00424, #X00672                              ______________________________________                                    

II. PRIMER OLIGONUCLEOTIDES

Primer oligonucleotides were synthesized by Genosys Biotechnologies,Inc. (Woodlands, Tex., USA), Integrated DNA Technologies, Inc.(Coralville, Iowa, USA), Genemed Biotechnologies, Inc. (San Francisco,Calif., USA), and Cruachem, Inc. (Dulles, Va., USA). The original primeroligonucleotides were extensively modified to avoid cross-reactivity andto ensure non-preferential amplification. Presently preferred optimizedprimers are described in Tables 5 and 6.

                  TABLE 5                                                         ______________________________________                                        SELECTED PRIMER CHARACTERISTICS                                                     Primer                     Melting Product                              SEQ   Designa-           Map     Temperature                                                                           Length                               ID NO:                                                                              tion     Region    Position                                                                              (°C.)                                                                          (bp)                                 ______________________________________                                        1     SAL-1.4  invE gene.sup.1                                                                         18-37   59      469                                  2     SAL-2.4  invA gene.sup.1                                                                         487-465 59                                           3     virF-1.3 virF locus of                                                                           623-646 59      215                                  4     virF-2.1 virulence 838-816 59                                                          plasmid.sup.2                                                  5     CFO3R.2  flagellin 1746-1171                                                                             60       340-                                6     CFO4R    gene.sup.3                                                                              2113-2090                                                                             60      360                                  7     YE 1.1   ail gene.sup.4                                                                          547-566 59      287                                  8     YE 2               834-815 59                                           9     ECO1.1   uidA gene.sup.5                                                                         198-217 59      423                                  10    ECO 2              621-601 60                                           11    βglob-1                                                                           human     54-73   59      262                                  12    βglob-2.1                                                                         gene.sup.6                                                                              -195/-176                                                                             59                                           ______________________________________                                         .sup.1 Stone, G., Oberst, R, Hays, M, McVey, S., Chengappa, M., Detection     of Salmonella Serovars from Clinical Samples by Enrichment Broth              CultivationPCR Procedure, J. Clin. Microbiol.; 32: 1742-1749 (1994).          .sup.2 Yavzori, M., Cohen, D., Wasserlauf, R., Ambar, R., Rechavi, G.,        Ashkenazi, S., Identification of Shigella Species in Stool Specimens by       DNA Amplification of Different Loci of the Shigella Virulence Plasmid,        Eur. J. Clin. Microbiol. Infect. Dis.; 13: 232-237 (1994).                    .sup.3 Wegmuller, B., Luthy, J., Candrian, U., Direct Polymerase Chain        Reaction Detection of Campylobacter jejuni and Campylobacter coli in Raw      Milk and Dairy Products, Applied ad Environmental Microbiology; 59:           2161-2165 (1993).                                                             .sup.4 Nakajima, H., Inoue, M., Mori, T., Itoh, K., Arakawa, E., Watanabe     H., Detection and Identification of Yersinia pseudotuberculosis and           Pathogenic Yersinia enterocolitica by Improved Polymerase Chain Reaction      Method, J. Clin. Microbiol.; 30: 2484-2486 (1992).                            .sup.5 Cebula, T., Payne, W., Feng, P., Simultaneous Identification of        Strains of Escherichia coli Serotype O157:H7 and Their ShigaLike Toxin        Type by Mismatch Amplification Mutation AssayMultiplex PCR, J. Clin.          Microbiol.; 33: 248-250 (1995).                                               .sup.6 Bauer, H., Ting, Y., Greer, C., Chambers, J., Tashiro, C., Chimera     J., Reingold, A., Monos, M., Genital Human Papillomavirus Infection in        Female University Students and Determined by a PCRBased Method, JAMA; 265     472-477 (1991).                                                          

                  TABLE 6                                                         ______________________________________                                        PREFERRED PRIMER SEQUENCES                                                    SEQ ID NO:  MODIFIED PRIMER SEQUENCE                                          ______________________________________                                        1           5'-TGAAATGGCAGAACAGCGTC-3'                                        2           5'-CGTTCTGAACCTTTGGTAATAAC-3'                                     3           5'-GATGAGGAAGCTTTATATACTTCG-3'                                    4           5'-TGCATGATGCATGCGAATATCAA-3'                                     5           5'-CAAAGTGGTTCTTATGCAATGGC-3'                                     6           5'-TGCTGCTGAGTTAATTCTAAGACC-3'                                    7           5'-TGTGTACGCTGCGAGTGAAA-3'                                        8           5'-GCCCCCAGTAATCCATAAAG-3'                                        9           5'-CTCTTCCATGGGTTTCTCAC-3'                                        10          5'-GATGCTCCATCACTTCCTGAT-3'                                       11          5'-CAACTTCATCCACGTTCACC-3'                                        12          5'-AAGAGCCAAGGACAGGTAC-3'                                         ______________________________________                                    

III. PROBE OLIGONUCLEOTIDES

Internal probe oligonucleotides, complementary to one strand of theamplification products of the PCR reactions performed with the primerslisted in Table 6, were obtained from Genosys Biotechnologies, Inc.(Woodlands, Tex., USA), Integrated DNA Technologies, Inc. (Coralville,Iowa, USA), Genemed Biotechnologies, Inc. (San Francisco, Calif., USA),and Cruachem, Inc. (Dulles, Va., USA). The probe designations andsequences are listed in Table 7.

                  TABLE 7                                                         ______________________________________                                        PROBE SEQUENCES                                                               SEQ            Probe                                                          ID             Desig-                                                         NO:  Analyte   nation  Probe Sequence                                         ______________________________________                                        13   Salmonella                                                                              SAL     5'-TGGTTGATTTCCTGATCGC-3'                                             P.1                                                            14   Shigella  SHIG    5'-CTGATCAGATAAGGAAGATTG-3'                                           P2.1                                                           15   Campylo-  CAMP    5'-AAACTTGGAACACTTCTTGCT-3'                                 bacter    P.1                                                            16   Yersinia  YE P    5'-GGCAGTAATAAGTTTGGTCAT-3'                            17   Escherichia                                                                             ECO P1  5'-GGAATTGATCAGCGTTGG-3'                               18   β-globin                                                                           βglob                                                                            5'-CACAACTGTGTTCACTAGC-3'                                             P.1                                                            ______________________________________                                    

IV. CLINICAL SAMPLES

Clinical stool samples were prepared in accord with the rapid sampleprocessing means described above.

V. PCR AMPLIFICATION

For amplification, a 50 μl reaction mixture was prepared using 5 μl of10 X PCR buffer (Promega Corp., Madison, Wis., USA), 1 μl (5.0 U/μl) Taqpolymerase, 1 μl (10 mM) each dNTP (Promega Corp., Madison, Wis., USA),5 μl of mixed primers (890 ng total primer concentration as describedbelow, see Table 8), 30 μl molecular grade water, and 5 μl (200 ng orless total DNA concentration) of ATCC strain DNA or clinical stoolsample DNA. Due to the variability of β-globin DNA in clinical stoolsamples, control DNA was extracted from human cell lines (MOLT-4, HFF)and added with the primer mixture to each amplification reaction at aconcentration of 0.1 μg/reaction.

PCR was performed using a Perkin Elmer 9600 thermocycler (Norwalk,Conn., USA) with one denaturing cycle for 3 minutes at 96° C. followedby 35 cycles as follows: 30 seconds at 94° C. for denaturing, 20 secondsat 61° C. for annealing, 20 seconds at 72° C. for extension.

VI. GEL DETECTION

PCR products were visualized (and confirmed by size) by ethidium bromidestaining after electrophoresis in 4% Nu Sieve 3:1 agar (FMC, Rockland,Md., USA). A 50-1000 bp ladder (Bio Ventures, Inc., Murfreesboro, Tenn.,USA) was used as a molecular size marker.

VII. COVALENT ATTACHMENT OF NUCLEIC ACID PROBES

All probes were synthesized with a 5' terminal primary amine.N-oxysuccinimide amine binding microtiter plates (Corning CostarCorporation, Cambridge, Mass., USA) were used as the solid phaseattachment support for the nucleic acid probes. Each probe was dilutedto 1 μg/ml in phosphate buffered saline (PBS), pH 9.0, and 100 μl ofeach probe was placed in a separate well. The plate was incubated for 1hour at room temperature. After incubation, the solution was aspiratedand the plate washed 5 times with 300 μl wash buffer/well (2.0 mMImidizole buffered saline, 0.02% Tween-20)(all chemicals obtained fromSigma Chemical Co., St. Louis, Mo., USA). The remaining active siteswere blocked with Stabilcoat (BSI Corporation, Eden Prairie, Minn., USA)for 30 minutes at room temperature. The wells were aspirated and useddirectly for hybridization.

VIII. DNA HYBRIDIZATION/COLORIMETRIC DETECTION

Amplified products (1×10¹² molecules) were diluted in 25 μl of PBS, pH7.25, and added with 25 μl denaturing solution (0.8N NaOH) to aprobe-coated well. After incubation for 10 minutes at room temperature,25 μl 4× hybridization solution (PBS, pH 7.25, 8% BSA) and 25 μlneutralizing solution (4M Ammonium Acetate)(Sigma Chemical Co., St.Louis, Mo., USA) were added and incubated for a further 5 minutes atroom temperature. The plate was then incubated for 45 minutes at 55° C.Following incubation, the plate was washed 5 times with wash buffer (1MTris-buffered saline, pH 7.5, 1.0% Tween 20) at room temperature.

For colorimetric analysis of the hybridized products bound to the wells,100 μl of streptavidin-alkaline phosphatase conjugate (SPA, Milan,Italy) was added and incubated for 30 minutes at 37° C. The plate waswashed 5 times with wash buffer and incubated a further 30 minutes at37° C. in 100 μl p-NPP solution (1 mg/ml p-nitrophenyl phosphate in 0.5MTris, pH 9.5). The reaction was stopped with the addition of 1.5N NaOH.Absorbance values were determined by analysis at 405 nm on a SLTLabinstruments Model #16-886 microplate reader (SLT, Salzburg, Austria).

RESULTS PRIMER OPTIMIZATION

Oligonucleotide primers were designed to amplify all 5 analytes andβ-globin internal control in a single PCR reaction tube. An analysis andmodification of the original primers was undertaken with the objectivesof improving the amplification efficiency of each primer and decreasingthe possibility of cross-reactivity among the primer pairs. Severalparameters were manipulated in order to minimize physical propertydifferences among the primers. Each primer was modified to approximatelythe same length, i.e., 19-24 bp. Primer oligonucleotides of this lengthresult in greater specificity in the amplification reaction whileshorter primers may result in the amplification of non-specificproducts. Because the efficiency of the primer pairs is also effected bythe presence of hairpin loops and dimers, OLIGO 5.0 software (NBI,Plymouth, Minn., USA) was used to analyze potential primers. If hairpinloops or dimers were found, the primer sequence was modified to removethem or to, at least, diminish the effect.

Cross-reactivity in a multiple primer reaction could also result indiminished amplification efficiency or in false positive amplifications.To analyze for cross-reactivity, a sequence alignment for each primerwas performed using a BioSCAN database search of the Genbank. Thecomputation was performed at the University of North Carolina, ChapelHill, N.C., USA, using the BioSCAN network server. No significantsequence matches were reported. To further test for cross-reactivity,each primer pair was amplified with DNA from each of the six analytes.It was found that the Salmonella primers would amplify non-specificproducts in a reaction with Molt 4 or HFF DNA, which were sources ofβ-globin DNA. To eliminate the cross-reactivity, new Salmonella primerswere created by shifting the sequence several bases upstream ordownstream from the original primer sequence. This shifting eliminatedthe cross-reactivity between the primers and the β-globin DNA.

In order to ensure that the primers are simultaneously annealing, themelting temperatures (Tm) need to be similar. Melting temperature foreach primer was determined using the Physical Property Predictionsoftware (Synthetic Genetics, Inc.) and the sequence was modified toobtain melting temperatures of 59°-60° C.

Preferential amplification in a multiple primer PCR process may occurdue to competition for limited reagents in the reaction as well asefficiency of each primer to anneal to the target nucleic acid sequence.Since the reaction dynamics are different for each combination ofprimers, the optimum conditions need to be determined empirically. Thesix analyte simultaneous PCR process was optimized for magnesiumconcentration (MgCl₂ concentration in the buffer solution), Taqpolymerase enzyme concentration, annealing temperature and primerconcentration. PCR and gel detection was performed as each of theseparameters were varied. Taq DNA polymerase was titrated from 2.0 U-5.5U/reaction and the optimum concentration for this primer mix wasdetermined to be 5.0 U/reaction. Magnesium concentrations were titratedfrom 1.0 mM to 6.0 MM and an optimum concentration of 1.5 mM wasdetermined. Amplification annealing temperatures tested ranged from 53°C. to 63° C. Under these conditions, optimum amplification efficiencyfor all six analytes was achieved at 61° C. Primer concentration wasmodified for each primer pair in order to obtain equal efficiency. Underthese selected optimum conditions, a preferred concentration of themodified primer pairs (SEQ ID NOS: 1-12) for each PCR reaction is shownin Table 8.

                  TABLE 8                                                         ______________________________________                                        A PREFERRED CONCENTRATION OF PRIMER PAIRS                                     SEQ ID                                                                              Primer Pair   Concentration (ng) of each Primer/each                    NOS:  Designations  Primer Pair                                               ______________________________________                                        1, 2  Sal 1.4, Sal 2.4                                                                            200/400                                                   3, 4  virF 1.3, virF 2.1                                                                          30/60                                                     5, 6  CFO3R.2, CFO4R                                                                               50/100                                                   7, 8  YE 1.1, YE 2   75/150                                                   9, 10 ECO 1.1, ECO 2                                                                              40/80                                                     11, 12                                                                              β glob 1, βglob 2.1                                                                50/100                                                   ______________________________________                                    

Each primer pair was amplified individually, with 200 ng ATCC strain DNA(Table 3) and 200 ng of appropriate primer, to confirm amplification ofthe expected fragment size. For simultaneous multiple amplifications,200 ng total concentration (40.0 ng of each bacterial analyte) of ATCCstrain DNA, 0.1 μg of control (β globin) DNA, and 890 ng totalconcentration of primer mixture (Table 8) was used as the positivecontrol. Clinical stool samples were amplified with 200 ng total(sample) DNA concentration, 0.1 μg of control (β globin) DNA, and 890 ngtotal primer mixture concentration (Table 8).

3. MULTIANALYTE RECOGNITION PROCESS MEANS/NUCLEIC ACID SEQUENCE MISMATCHDETECTION MEANS

As reviewed in the background section, numerous methods for detectingand discriminating nucleic acid sequences using oligonucleotide probes,i.e., probes complementary to the PCR-amplified products, are known.Either the probes or the PCR products are labeled with some type oflabel moiety so as to be detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. Examples of labelmoieties include fluorescent dyes, electron-dense reagents, enzymescapable of depositing insoluble reaction products or of being detectedchromogenically, such as horseradish peroxidase or alkaline phosphatase,a radioactive label such as ³² P, or biotin. The probes and PCR productsare mixed under hybridization conditions such that hybridization occursbetween the PCR products and probes which are sufficiently complementaryto each other. After hybridization, processing to remove anynon-hybridized molecules is performed such that detection of remaininglabeled component indicates the presence of probe/target nucleic acidhybrids.

Typically, a solid phase system such as, for example, a microtiter platehaving probe-coated wells, is used. In solid phase systems, an importantfactor affecting the efficiency of the detection process is theefficiency of the binding method used to immobilize the desiredcomponent, either the probe or the PCR products, onto the solid matrix.The use of covalent linking chemistry has recently been shown to producemore consistent and efficient binding of probe oligonucleotides tomicroplate plastic surfaces. In this technology, the oligonucleotideprobes are attached covalently to chemical linkers on the plasticsurface via a reactive moiety, such as an amine or phosphate group,attached to the 5'-end of the probe.

Also as reviewed in the background section, for some purposes, it isnecessary to differentiate minor differences between amplified nucleicacid sequences such as nucleotide substitutions, insertions ordeletions. These nucleotide variations may be mutant or polymorphicallele variations. Of particular interest and difficulty is thediscrimination of single-base mismatched nucleic acid sequences.Sequence-specific oligonucleotide probes, i.e., probes which are exactlycomplementary to an appropriate region of the target nucleic acidsequence, are typically used. All primers and probes, however, hybridizeto both exactly complementary nucleic acid sequence regions as well asto sequences which are sufficiently, but not exactly, complementary,i.e., regions which contain at least one mismatched base. Thus, aspecific probe will hybridize with the exact target nucleic acidsequence as well as any substantially similar but non-target nucleicacid sequences which are also present following the amplificationprocess.

Various approaches to discriminating these similar hybrids from oneanother have been used including, for example, stringent washingconditions and/or processing with toxic chemicals to affect the physicalproperties of the hybrid complexes. These known methods for detectingnucleic acid sequence base mismatches, however, are generally toodifficult, harsh, and inconvenient for routine laboratory use.

The multianalyte recognition process of the present invention isperformed with appropriate probe oligonucleotides and is preferablyperformed on microtiter plates incorporating covalent linking technologyto enhance the binding efficiency of the probe/nucleic acid sequencehybrids. In addition, nucleic acid sequence mismatch detection means areprovided to permit discrimination between amplified nucleic acidsequences having minor mismatches, including only single basemismatches. The nucleic acid sequence mismatch detection means utilizesequence-specific probe oligonucleotides incorporating neutral basesubstitution molecules, strategically positioned to discriminate thebase differences of interest, to reduce the strength of thehybridization between any mismatched nucleic acid sequences and theprobe. Because the mismatched sequence hybrids are thereby madesignificantly weaker than the matched sequence hybrids, differentiationof matched and mismatched hybrids is possible without the imposition ofstringent or harsh processing conditions.

With respect to the gastroenteritis panel application, it is critical tobe able to detect the presence of a highly virulent enterohemorrhagicstrain of E. coli known as O157:H7. This virulent strain differs fromother E. coli strains, represented by E. coli O25:K98:NM, by only asingle base pair in the uidA gene. Because of the substantial similarityin nucleic acid sequences, both of these strains, if present, willamplify during a PCR amplification process using primers to target anucleic acid sequence including the uidA gene. Thus, it is necessary tobe able to distinguish whether the virulent O157:H7 strain is present inthe PCR amplification products.

In accord with the present invention, this problem is solved through theuse of modified sequence-specific probes which incorporate neutral basesubstitution molecules. In particular, as illustrated in Example 4, atleast one inosine molecule is substituted, at a position other than thealready present single-base mismatch, within probes otherwisesequence-specific for E. coli O157:H7. These modified probes ensure thatthe probe/O157:H7 strain nucleic acid sequence hybrids will matchcompletely, except at the inosine location(s), while theprobe/O25:K98:NM strain nucleic acid sequence hybrids will additionallymismatch at the single base mismatch location. Thus, theprobe/O25:K98:NM strain nucleic acid sequence hybrids are made weaker tothereby be sufficiently removable, without the use of harsh andinconvenient processing conditions or the use of toxic chemicals, topermit accurate discrimination between the single-base mismatched E.coli strains.

EXAMPLE 4 MATERIALS AND METHODS I. OLIGONUCLEOTIDE PROBES

Nucleic acid probes were synthesized by Genosys Biotechnologies, Inc(The Woodlands, Tex., USA). The single-base mismatched sequence-specificprobes for the O25:K98:NM E. coli strain and for the virulent O157:H7 E.coli strain are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        SEQUENCE-SPECIFIC PROBES FOR TWO STRAINS OF E. coli                           SEQ  Probe   E. coli                                                          ID   Desig-  Strain                                                           NO:  nation  Specificity                                                                             Probe Sequence                                         ______________________________________                                        19   M6      O25:K98:  5'- GGA ATT GAT* CAG CGT TGG-3'                                     NM                                                               20   M7      O157:H7   5'-T GGA ATT GAG* CAG CGT TG-3'                        ______________________________________                                         *single-base mismatch location                                           

The sequences of the modified probes having at least one inosine (i)substitution are shown in Table 10.

                  TABLE 10                                                        ______________________________________                                        MODIFIED E. coli O157:H7 SEQUENCE-SPECIFIC PROBES                             SEQ    Probe                                                                  ID NO: Designation                                                                              Probe Sequence                                              ______________________________________                                        21     M1         5'- GT GGA ATT iAG CAG CGT TG-3'                            22     M2         5'- GT GGA ATT GAG iAG CGT TG-3'                            23     M3         5'- GT GGA ATT GAG CAi CGT TG-3'                            24     M4         5'-TGT GiA ATT iAG CAi CGT TGG T-3'                         25     M5         5'-TGT GGA ATT iAG iAi CGT TGG T-3'                         26     M8         5'- GGA ATT iAG CAG CGT TGG-3'                              27     M9         5'- GGA ATT GAG iAG CGT TGG-3'                              28     M10        5'- GGA ATT iAG iAG CGT TGG-3'                              29     M11        5'- TGT GGA ATT GAG iAG CGT TG-3'                           30     M12        5'- GT GGA ATT GAG iAG CGT TGG-3'                           ______________________________________                                    

II. COVALENT ATTACHMENT OF NUCLEIC ACID PROBES

All probes were synthesized with a 5' terminal primary amine.N-oxysuccinimide amine binding microtiter plates (Corning CostarCorporation, Cambridge, Mass., USA) were used as the solid phaseattachment support for the nucleic acid probes. Each probe was dilutedto 1 μg/nl in phosphate buffered saline (PBS), pH 9.0, and 100 μl ofeach probe was placed in a separate well. The plate was incubated for 1hour at room temperature. After incubation, the solution was aspiratedand the plate washed 5 times with 300 μl wash buffer/well (2.0 mMImidizole buffered saline, 0.02% Tween-20)(all chemicals obtained fromSigma Chemical Co., St. Louis, Mo., USA). The remaining active siteswere blocked with Stabilcoat (BSI Corporation, Eden Prairie, Minn., USA)for 30 minutes at room-temperature. The wells were aspirated and useddirectly for hybridization.

III. PCR AMPLIFICATION

For amplification, a 50 μl reaction mixture was prepared using 5 μl of10 X PCR buffer (Promega Corp., Madison, Wis., USA), 0.5 units Taq DNApolymerase, 200 μM each dNTP (deoxynucleotide triphosphate)(PromegaCorp., Madison, Wis., USA), 200 ng of each primer specific for the uidAgene of E. coli (SEQ ID NOS: 9 and 10, see Table 6), and 200 ng ofeither E. coli O25:K98:NM (ATCC #43886, See Table 3) DNA or E. coliO157:H7 (ATCC #35150, see Table 3) DNA. One primer, SEQ ID NO:10, wasbiotinylated at the 5' terminal end.

PCR was performed using a Perkin Elmer 9600 thermocycler (Norwalk,Conn., USA) with one denaturing cycle for 3 minutes at 96° C. followedby 35 cycles as follows: 30 seconds at 94° C. for denaturing, 20 secondsat 61° C. for annealing, 20 seconds at 72° C. for extension.

IV. GEL DETECTION

PCR products were visualized (and confirmed by size) by ethidium bromidestaining after electrophoresis in 4% Nu Sieve 3:1 agar (FMC, Rockland,Md., USA). A 50-1000 bp ladder (Bio Ventures, Inc., Murfreesboro, Tenn.,USA) was used as a molecular size marker.

V. DNA HYBRIDIZATION/COLORIMETRIC DETECTION

Each of the amplified products (1×10¹² molecules) was diluted in 25 μlof PBS, pH 7.25, and added with 25 μl denaturing solution (0.8N NaOH) toa probe-coated well. After incubation for 10 minutes at roomtemperature, 25 μl 4× hybridization solution (PBS, pH 7.25, 8% BSA) and25 μl neutralizing solution (4M Ammonium Acetate)(Sigma Chemical Co.,St. Louis, Mo., USA) were added and incubated for a further 5 minutes atroom temperature. The plate was then incubated for 45 minutes at 55° C.Following incubation, the plate was washed 5 times with wash buffer (1MTris-buffered saline, pH 7.5, 1.0% Tween 20) at room temperature. Forcalorimetric analysis of the hybridized products bound to the wells, 100μl of streptavidin-alkaline phosphatase conjugate (SPA, Milan, Italy)was added and incubated for 30 minutes at 37° C. The plate was washed 5times with wash buffer and incubated a further 30 minutes at 37° C. in100 μl p-NPP solution (1 mg/ml p-nitrophenyl phosphate in 0.5M Tris, pH9.5). The reaction was stopped with the addition of 1.5N NaOH.Absorbance values were determined by analysis at 405 nm on a SLTLabinstruments Model #16-886 microplate reader (SLT, Salzburg, Austria).

RESULTS

As seen in Table 11, the modified probes used to discriminate betweenthe uidA allele of the O157:H7 E. coli and the O25:K98:NM E. colistrains substitute inosine molecules for guanosine and cytosine invarying locations. Because guanosine and cytosine affect strong triplebonds, substitution of these bases with the neutral base, inosine, wasanticipated to have the most effect. The probes were designed by varyingthe placement and number of inosines in a given DNA sequence. The probelengths were also modified to maximize the discrimination.

As described above, each probe was covalently linked to a 96-wellmicrotiter plate and then used immediately for hybridization assays. Tominimize the possible variability of covalent linking of the probes tothe wells, the assays were run in triplicate on each plate and a totalof six plates were used for the hybridization studies. The DNA used forhybridization was amplified from either E. coli O157:H7 or E. coliO25:K98:NM. The 423 bp region amplified corresponded to nucleotides198-621 of the uidA gene. The amplified DNA sequences were analyzed bymigration on a 4% agarose gel. Serial dilutions of the products werequantitated by comparison with a known standard on the gel. The plateswere hybridized with equal amounts of amplificationproducts(approximately 1×10¹² molecules/well). Time course experimentsdetermined that the hybridization signal reached saturation by 45minutes at 55° C. Accordingly, all experiments were analyzed byhybridization at 55° C. for 45 minutes.

Two parameters were determined to judge the efficacy of discriminationof a given probe. The ratio of O157:H7 signal to O25:K98:NM signal wasdetermined for each probe to establish the discrimination of the probes.The signal achieved with the sequence specific O157:H7 probe (100%), SEQID NO:20, was compared to the O157:H7 signal retained by each probe anda percentage value calculated to establish the sensitivity of each probeas a marker for identification of the pathogenic strain. The results ofthese determinations, calculated for all six experiments, are shown inTable 11.

                  TABLE 11                                                        ______________________________________                                        EXPERIMENTAL RESULTS                                                                Probe                                                                   SEQ   Designa- Ratio of O157:H7 to                                                                           Percentage of                                  ID NO:                                                                              tion     O125:K98:NM absorbance                                                                        O157:H7 absorbance                             ______________________________________                                        19    M6       0.45 +/- 0.19   32                                             20    M7       2.19 +/- 0.11   100                                            21    M1       3.17 +/- 0.37   86                                             22    M2       5.26 +/- 2.44   46                                             23    M3       2.56 +/- 0.59   66                                             24    M4       2.58 +/- 0.70   82                                             25    M5       3.09 +/- 0.92   27                                             26    M8       3.55 +/- 0.73   63                                             27    M9       5.52 +/- 2.69   49                                             28    M10      3.26 +/- 2.25   24                                             29    M11      3.05 +/- 0.61   73                                             30    M12      2.84 +/- 0.32   75                                             ______________________________________                                    

Based on the selected parameters, the M1 probe exhibits the bestdiscrimination (3.17+/-0.37) while retaining the best signal (86%).Other probes show excellent discrimination, e.g., probes M2, M8 and M9,however, the variability is increased and the signal is reduced.Substitution of inosine at position 11 and 13 (probes M2, M9, M11, M12,and M3, respectively) demonstrated highly variable or reduceddiscrimination and a reduced overall signal intensity. Probes M2 and M9have an inosine substitution immediately downstream to the single basemismatch and both demonstrated reduced signal intensity and highvariability of discrimination. The variability was reduced and signalintensity was partially recovered by increasing the length of theprobes, e.g., probes M11 and M12. Probe M3, with an inosine substitutionat position 13, demonstrated less of an effect on signal intensity butthe discrimination was reduced. Substitution of multiple inosines, as inprobes M4, M5, and M10, resulted in good discrimination but a reducedsignal. Comparisons of probes M9 and M2 to M11 and M12 and comparison ofprobe M8 to M1 indicates that increasing the length of the probeincreases the signal intensity while also stabilizing the variability ofthe discrimination.

It will be appreciated from the above results that the incorporation ofneutral base substitution into sequence-specific probes achieves asensitivity and efficiency capable of differentiating even single basemismatches between amplified nucleic acid sequences.

The present invention may be embodied or utilized in other specificforms or manners without departing from its spirit or essentialcharacteristics. The described embodiments and methods are to beconsidered in all respects only as illustrative and not restrictive. Thescope of the invention is, therefore, indicated by the appended claimsrather than by the foregoing description. All changes which come withinthe meaning and range of equivalency of the claims are to be embracedwithin their scope.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 30                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TGAAATGGCAGAACAGCGTC20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGTTCTGAACCTTTGGTAATAAC23                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GATGAGGAAGCTTTATATACTTCG24                                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TGCATGATGCATGCGAATATCAA23                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CAAAGTGGTTCTTATGCAATGGC23                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TGCTGCTGAGTTAATTCTAAGACC24                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TGTGTACGCTGCGAGTGAAA20                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GCCCCCAGTAATCCATAAAG20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CTCTTCCATGGGTTTCTCAC20                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GATGCTCCATCACTTCCTGAT21                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CAACTTCATCCACGTTCACC20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AAGAGCCAAGGACAGGTAC19                                                         (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TGGTTGATTTCCTGATCGC19                                                         (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CTGATCAGATAAGGAAGATTG21                                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AAACTTGGAACACTTCTTGCT21                                                       (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGCAGTAATAAGTTTGGTCAT21                                                       (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GGAATTGATCAGCGTTGG18                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CACAACTGTGTTCACTAGC19                                                         (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GGAATTGATCAGCGTTGG18                                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TGGAATTGAGCAGCGTTG18                                                          (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GTGGAATTNAGCAGCGTTG19                                                         (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GTGGAATTGAGNAGCGTTG19                                                         (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GTGGAATTGAGCANCGTTG19                                                         (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TGTGNAATTNAGCANCGTTGGT22                                                      (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      TGTGGAATTNAGNANCGTTGGT22                                                      (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GGAATTNAGCAGCGTTGG18                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GGAATTGAGNAGCGTTGG18                                                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GGAATTNAGNAGCGTTGG18                                                          (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TGTGGAATTGAGNAGCGTTG20                                                        (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: N = INOSINE                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GTGGAATTGAGNAGCGTTGG20                                                        __________________________________________________________________________

What is claimed and desired to be secured by United States LettersPatent is:
 1. A primer pair for non-preferentially amplifying, withminimal cross-reactivity, nucleic acid sequences from microorganisms ofthe species Salmonella, when present, in a mixture of analytes within aclinical sample, said primer pair consisting of SEQ. ID. NOS. 1 and 2.2. A primer pair for non-preferentially amplifying, with minimalcross-reactivity, target nucleic acid sequences from microorganisms ofthe species Yersinia, when present, in a mixture of analytes within aclinical sample, said primer pair consisting of SEQ. ID. NOS. 7 and 8.3. A probe for use in detecting target nucleic acid sequences frommicroorganisms of the species Yersinia, amplified by the primer pairconsisting of SEQ. ID. NOS. 7 and 8, said probe consisting of SEQ. ID.NO.
 16. 4. A primer pair for non-preferentially amplifying, with minimalcross-reactivity, target nucleic acid sequences from E. colimicroorganisms, when present, in a mixture of analytes within a clinicalsample, said primer pair consisting of SEQ. ID. NOS. 9 and
 10. 5. Acomposition consisting of a plurality of primer pairs for simultaneouslynon-preferentially amplifying, with minimal cross-reactivity, multipletarget nucleic acid sequences from a plurality of organisms, whenpresent, in a mixture of nucleic acid sequences, said plurality ofprimer pairs consisting of two or more primer pairs selected from thegroup consisting of SEQ. ID. NOS. 1 and 2, SEQ. ID. NOS. 3 and 4, SEQ.ID. NOS. 5 and 6, SEQ. ID. NOS. 7 and 8, and SEQ. ID. NOS. 9 and
 10. 6.A composition consisting of a plurality of primer pairs forsimultaneously non-preferentially amplifying, with minimalcross-reactivity, multiple target nucleic acid sequences from aplurality of organisms, if present, in a mixture of nucleic acidsequences, said plurality of primer pairs including at least one primerpair selected from the group consisting of SEQ. ID. NOS. 1 and 2, SEQ.ID. NOS. 7 and 8, and SEQ. ID. NOS. 9 and
 10. 7. A composition asdescribed in claim 6 further comprising a primer pair consisting of SEQ.ID. NOS. 3 and
 4. 8. A composition as described in claim 6 furthercomprising a primer pair consisting of SEQ. ID. NOS. 5 and
 6. 9. Acomposition consisting of a plurality of probes for discriminatingmultiple target nucleic acid sequences from a plurality of organisms, ifpresent, within a mixture of amplified nucleic acid sequences, saidplurality of probes consisting of two or more probes selected from thegroup consisting of SEQ. ID. NOS. 13 through
 17. 10. A compositionconsisting of a plurality of probes for discriminating multiple targetnucleic acid sequences from a plurality of organisms, if present, withina mixture of amplified nucleic acid sequences, said plurality of probesincluding SEQ. ID. NO.
 16. 11. A composition as described in claim 10further comprising a probe consisting of SEQ. ID. NO.
 13. 12. Acomposition as described in claim 10 further comprising a probeconsisting of SEQ. ID. NO.
 14. 13. A composition as described in claim10 further comprising a probe consisting of SEQ. ID. NO.
 15. 14. Acomposition as described in claim 10 further comprising a probeconsisting of SEQ. ID. NO. 17.